Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.

Two-Photon Excitation Fluorescence Microscopy

Here we report that aloe-emodin (AE), a hydroxyanthraquinone present in Aloe vera leaves, has a specific in vitro and in vivo antineuroectodermal tumor activity. The growth of human neuroectodermal tumors is inhibited in mice with severe combined immunodeficiency without any appreciable toxic effects on the animals. The compound does not inhibit the proliferation of normal fibroblasts nor that of hemopoietic progenitor cells. The cytotoxicity mechanism consists of the induction of apoptosis, whereas the selectivity against neuroectodermal tumor cells is founded on a specific energy-dependent pathway of drug incorporation. Taking into account its unique cytotoxicity profile and mode of action, AE might represent a conceptually new lead antitumor drug.

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Aloe-emodin Is a New Type of Anticancer Agent with Selective Activity against Neuroectodermal Tumors

Linear Inverse Problems.- Large-Scale Inverse Problems in Imaging.- Regularization Methods for Ill-Posed Problems.- Distance Measures and Applications to Multi-Modal Variational Imaging.- Energy Minimization Methods.- Compressive Sensing.- Duality and Convex Programming.- EM Algorithms.- Iterative Solution Methods.- Level Set Methods for Structural Inversion and Image Reconstructions.- Expansion Methods.- Sampling Methods.- Inverse Scattering.- Electrical Impedance Tomography.- Synthetic Aperture Radar Imaging.- Tomography.- Optical Imaging.- Photoacoustic and Thermoacoustic Tomography: Image Formation Principles.- Mathematics of Photoacoustic and Thermoacoustic Tomography.- Wave Phenomena.- Statistical Methods in Imaging.- Supervised Learning by Support Vector Machines.- Total Variation in Imaging.- Numerical Methods and Applications in Total Variation Image Restoration.- Mumford and Shah Model and its Applications in Total Variation Image Restoration.- Local Smoothing Neighbourhood Filters.- Neighbourhood Filters and the Recovery of 3D Information.- Splines and Multiresolution Analysis.- Gabor Analysis for Imaging.- Shaper Spaces.- Variational Methods in Shape Analysis.- Manifold Intrinsic Similarity.- Image Segmentation with Shape Priors: Explicit Versus Implicit Representations.- Starlet Transform in Astronomical Data Processing.- Differential Methods for Multi-Dimensional Visual Data Analysis.- Wave fronts in Imaging, Quinto.- Ultrasound Tomography, Natterer.- Optical Flow, Schnoerr.- Morphology, Petros.- Maragos.- PDEs, Weickert. - Registration, Modersitzki. - Discrete Geometry in Imaging, Bobenko, Pottmann.-Visualization, Hege.- Fast Marching and Level Sets, Osher.- Couple Physics Imaging, Arridge.- Imaging in Random Media, Borcea.- Conformal Methods, Gu.- Texture, Peyre.- Graph Cuts, Darbon.- Imaging in Physics with Fourier Transform (i.e. Phase Retrieval e.g Dark field imaging), J. R. Fienup.- Electron Microscopy, Oktem Ozan.- Mathematical Imaging OCT (this is also FFT based), Mark E. Brezinski.- Spect, PET, Faukas, Louis.

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Handbook of mathematical methods in imaging

This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

Two-photon fluorescence excitation and related techniques in biological microscopy.

In iterative image restoration methods, implementation of efficient matrix vector multiplication, and linear system solves for preconditioners, can be a tedious and time consuming process. Different blurring functions and boundary conditions often require implementing different data structures and algorithms. A complex set of computational methods is needed, each likely having different input parameters and calling sequences. This paper describes a set of Matlab tools that hide these complicated implementation details. Combining the powerful scientific computing and graphics capabilities in Matlab, with the ability to do object-oriented programming and operator overloading, results in a set of classes that is easy to use, and easily extensible.

Iterative Methods for Image Deblurring: A Matlab Object-Oriented Approach

Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode.

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture

http://www.dmphotonics.com/Femtosecond_Imaging/Two-Photon_Pifoc.pdf

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

The adaptation of a confocal laser scanning microscope to a two-photon architecture for 3-D fluorescence microscopy is described.

Two-photon excitation imaging based on a compact scanning head

In our laboratory, within the framework of a national INFM project, we have realised a two-photon excitation fluorescence microscope part of a multipurpose architecture including also lifetime imaging and fluorescence correlation spectroscopy modules. The core of the architecture are a mode-locked Ti:sapphire laser (Tsunami 3960, Spectra Physics Inc., CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc., CA) and an ultracompact scanning head, Nikon PCM2000 (Nikon Instr., Italy). Three-dimensional confocal images of fluorophores excitable in UV and the blue region are shown. We are currently characterising the architecture in terms of point-spread-function and biological system viability.

http://ieeexplore.ieee.org/iel5/6513/17403/00804254.pdf

M. Corosu

Papers

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture

Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

Two-photon excitation imaging based on a compact scanning head

Three-dimensional fluorescence microscopy in two-photon excitation regime